A fundamental question in evolutionary genetics concerns the roles of mutational pleiotropy and epistasis in shaping trajectories of protein evolution. A powerful means of addressing this question involves the use of site-directed mutagenesis to explore the mutational landscape of protein function in experimentally defined regions of sequence space. Here we describe a plan to evaluate how pleiotropic trade-offs and epistatic interactions influence the selective accessibility of alternative mutational pathways during the adaptive functional evolution of mammalian hemoglobin (Hb). Using ancestral protein resurrection in conjunction with a combinatorial protein-engineering approach based on site-directed mutagenesis, we will examine the structural and functional effects of sequential mutational steps in all possible pathways that lead to the evolution of an increased Hb-O2 affinity in mammals that have adapted to environmental hypoxia. To evaluate the influence of pleiotropy and epistasis on adaptive protein evolution, we will examine the molecular basis of adaptive changes in Hb function at several different levels of divergence between pairs of mammalian taxa that have evolved different Hb-O2 affinities. The experimental approach integrates biochemical and biophysical examinations of the effects of specific mutations in recombinantly expressed Hbs. One of the primary innovations of this project is that we have developed an expression vector system that allows us to synthesize recombinant Hb in E. coli host cells. The research is designed to accomplish the following aims: (1) Identify the specific mutations that contribute to evolutionary changes in Hb function, and determine the relative contributions of additive and epistatic effects, and (2) Identify and characterize the biochemical/biophysical mechanisms responsible for observed pleiotropic effects and epistatic interactions. Accomplishing these two aims will reveal the specific mutations that have contributed to adaptive modifications of protein function, and will elucidate the specific biochemical/biophysical mechanisms by which pleiotropy and epistasis affect the selective accessibility of evolutionary pathways. By using Hb as a model molecule, we can leverage extremely rich sources of information about structure-function relationships to gain insights into mechanism. By directly measuring the structural and functional effects of causative mutations, our experimental results will provide answers to fundamental questions about molecular adaptation and mechanisms of protein evolution.